Versatile platform for genome-wide R-loop mapping for aging and neurodegenerative disease research

PROJECT SUMMARY R-loops are triple-stranded nucleic acid structures consisting of a DNA:RNA hybrid and a displaced single DNA strand. R-loops are generally transient in nature, but persistence of stable R-loops leads to DNA damage and genome instability, and dysregulation of these structures is strongly associated with aging and neurodegenerative diseases. As such, the study of R-loops is essential to understand mechanisms driving these diseases, and for the development of novel biomarkers and therapeutics. However, there remains much debate in the field regarding the specifics of how and where R-loops are formed, and how they are implicated in gene regulation in both normal and disease states. This is largely due to a lack of reliable approaches for mapping the genomic distribution of R-loops in vivo. While multiple approaches exist, many utilize an affinity purification strategy with an unreliable antibody (S9.6), requiring high inputs and generating noisy data with off-target artifacts. As such, to accelerate clinical research on neurodegenerative diseases, there is a strong market need for a next-generation R-loop mapping assay that is highly sensitive, scalable, and reliable. Here, EpiCypher is partnering with Dr. Jessica Tyler (Weill Cornell) to develop CUTANA-RH™, a novel approach for genome-wide profiling of R-loops to accelerate aging and neurodegenerative disease research. The first major innovation of this proposal is the development of a modular immunotethering strategy for genomic mapping of R-loops, which is compatible with both CUT&RUN- and CUT&Tag-based workflows. Rather than the unreliable S9.6 antibody, our approach leverages a catalytically inactive RNase H1 enzyme (RH∆) as a highly specific detection reagent for the DNA:RNA hybrid component of R-loops. In the CUTANA-RH workflows, cells or nuclei are incubated with GST-tagged RH∆ followed by an anti-GST antibody. Next, pAG-MNase (or pAG- Tn5) is added for targeted cleavage and sequencing of cleaved fragments, yielding precise localization of R-loops genome-wide. The second major innovation of our approach is the development of a designer nucleosome (dNuc) spike-in control panel including dNucs with RNA:DNA hybrid linkers (RH-dNucs). These will be utilized to 1) perform technical monitoring for assay optimization and sample quality control, and 2) normalize data for quantitative comparisons. The gains in throughput, sensitivity, and quantification provided by CUTANA- RH compared to affinity-based approaches will be highly enabling for basic and clinical research. In Phase I equivalent studies, we developed a first-generation CUTANA-RH assay and demonstrated its ability to reliably map R-loops in cultured cells. In Phase II, we will generate RH-dNucs and optimize the CUTANA-RH workflows (Aim 1). We will then validate our assays in a stimulation model and optimize protocols for primary cells and diverse tissues (Aim 2). We will prepare for commercialization by scaling manufacturing, validating CUTANA-RH beta-kits, and developing automated workflows to support services (Aim 3). Finally, we will demonstrate the power of this assay for clinical applications by profiling R-loops in Alzheimer’s disease brain tissue (Aim 3).